Ligand-directed targeting of malignant gliomas

The dramatic poor survival of patients diagnosed with glioblastoma multiforme (GBM) is a reflection of the struggles that accompany traditional treatments. Thus, the development of molecular-based targeted therapies represents new windows for intervention. In this study, we hypothesized that we could select peptide-ligands that selectively target GBM based on the idea that the glioma microenvironment may induce or modify the expression of cell surface receptors that could be accessed by circulating peptides. To select the peptides we employed two distinct in vivo screenings. First, a random phage-displayed peptide library was injected into mice bearing intracranial tumors. Phage that bound to tumor were recovered and sequenced. We found that the tumor-derived phage CLSYKGRC, CNKVSTKC and CQSSREKC were recovered with the highest frequencies and used for subsequent targeting experiments. Second, the phage peptide library was injected into mice without tumors and phage were recovered from brain and sequenced. A phage-displayed peptide (CRTIGPSVC) with homology to transferrin (Tf) was selected and injected into brain tumor-bearing mice. Results showed that after 6 hours of circulation, the CLSYKGRC, CNKVSTKC and CQSSREKC-phage selectively targeted GBM vasculature. In contrast, Tf-like phage accumulated outside the tumor blood vessels in the cytoplasm of cells located within GBM, suggesting it was internalized in vivo. However, after short periods of circulation this phage was restricted to the tumor vasculature. Importantly, none of the selected phage targeted normal brain cells in animals bearing intracranial tumors. An affinity column coupled to the CNKVSTKC zpeptide was used to identify receptors from GBM. Using mass-spectrometry Vimentin, a marker of glial malignancy, was identified as a potential receptor. Other studies showed that the Tf-like phage bound selectively to Apo-Tf (iron free), with no binding to Holo-Tf (iron loaded) or to Tf receptor (TfR). However, the binding of Tf-like phage to glioma cells that express TfR increased in the presence of Apo-Tf. Thus, the Tf-like phage could indirectly target TfR using the endogenous Tf pathway. We propose that the novel peptides identified in this study could be conjugated to therapeutic or imaging agents for use GBM. ^

Matrix metalloproteinase-1 as a prognosticator in malignant glioma

The overall purpose of this study was to assess the relationship between the promoter region polymorphism (-2607 1G/2G) of matrix metalloproteinase-1 (MMP-1) polymorphism and outcome in brain tumor patients diagnosed with a primary brain tumor between 1994 and 2000 at The University of Texas M. D. Anderson Cancer Center. The MMP-1 polymorphism was genotyped for all brain tumor patients who participated in the Family Brain Tumor Study and for whom blood samples were available. Relevant covariates were abstracted from medical records for all cases from the original protocol, including information on demographics, tumor histology, therapy and outcome was obtained. The hypothesis was that brain tumor patients with the 2G allele have a poorer prognosis and shorter survival than brain tumor patients with the 1G allele. ^ Experimental Design: Genetic variants for the MMP-1 enzyme were determined by a polymerase chain reaction-restriction fragment length polymorphism assay. Comparison was made between the overall survival for cases with the 2G polymorphism and overall survival for cases with the 1G polymorphism using multivariable Cox Proportional-Hazard analysis, controlling for age, sex, Karnofsky Performance Scale (KPS), extent of surgery, tumor histology and treatment received. Kaplan-Meier and Cox Proportional-Hazard analyses were utilized to assess if the MMP-1 polymorphisms were related to overall survival. Results: Overall survival was not statistically significantly different between the 2G allele brain tumor patients and the 1G allele patients and there was no statistically significant difference between tumor types. ^ Conclusions: No association was found between MMP-1 polymorphisms and survival in patients with malignant gliomas. ^

The role of deregulated E2F1 expression in brain tumors

Mutations disabling the retinoblastoma (Rb) pathway are among the most common in human cancers, including brain cancer. These mutations promote tumor development through deregulated control of the E2F family of transcription factors. E2F1 belongs to a class of E2F's identified as transcriptional activators and involved in the G1/S phase transition of the cell. However, E2F-1 presents with a paradox as it is considered to have membership in two gene classes, functioning as both an oncogene and a tumor suppressor. This unusual trait generates a degree of uncertainty on the role that E2F1 plays in the development or maintenance of any given tumor. Here we show that E2F1 functions as an oncogene in brain tumors through the generation of mice engineered to overexpress E2F1 specifically within glial cells and neuronal progenitors as directed by the GFAP promoter. Mice carrying the transgene develop with high penetrance a phenotype characterized by neurological deficits including paresia, ataxia, head tilt and seizures. MRI imagining of the tgE2F1 mice reveals a low incidence of mild hydrocephalus, and most notably, histological analysis demonstrates that 25% of tgE2F1 mice present with the spontaneous formation of malignant brain tumors. Overall these neoplasms show histological features from a wide range of aggressive brain cancers including medulloblastoma, choroid plexus carcinoma, primary neuroectodermic tumor and malignant gliomas. Isolation and characterization of astrocytes from the tgE2F1 animal reveals a highly proliferative population of cells with 55% ± 2.5 of the tgE2F1astrocytes, 35% ± 3.4 normal mouse astrocytes in S-phase and the acquired capacity to grow in anchorage independent conditions. Additionally tgE2F1 astrocytes show an aberrant phenotype with random chromosomal fusions and nearly all cells demonstrating polyploidy. Taken together, this model forces a comparison to human brain tumor formation. Mouse age as related to tumoral mimics the human scenario with juvenile tgE2F1 mice presenting embryonal tumors typically identified in children, and older tgE2F1 mice demonstrating gliomas. In this regard, this study suggests a global role for E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. ^

Modulation of radiation-induced genetic damage by HCMV: A glioma case-control study

Human cytomegalovirus (HCMV) infection occurs early in life and leads to life-long viral persistence. An association between HCMV infection and malignant gliomas has been reported suggesting that HCMV may play a role in glioma pathogenesis. The reported effects of HCMV on cells suggest that it could facilitate accrual of genotoxic damage. We therefore tested the hypothesis that HCMV infection modifies the sensitivity of cells to genetic damage from environmental insults such as γ-irradiation. Peripheral blood lymphocytes from 110 glioma patients and 100 controls were used to measure the level of both chromosome damage and cell death as endpoints for genetic instability. For each study participant, the extent of baseline, HCMV-, γ-radiation- and both – induced genetic instability was evaluated. Radiation induced a significant increase in aberration frequency over baseline in both cases and controls. Similarly, HCMV induced a significant increase in aberration frequency regardless of the disease status. Interestingly, HCMV induced damage was either equal or higher than that induced by radiation. Infected with HCMV prior to challenge with γ-radiation demonstrated a significant increase in the aberration frequency as compared to baseline, radiation- or HCMV-treated cells. With regards to apoptosis, cases showed a lower percentage of induction following in vitro exposure to γ-radiation and/or HCMV infection. The level of apoptosis was inversely related to the amount of chromosome damage in the cases, but not in the controls. These data indicate that, HCMV infection enhances the sensitivity of PBLs to γ-radiation-induced genetic damage.^

Molecular alterations in nucleotide excision repair genes in malignant glioma and their relevance to malignant progression and drug resistance

Recently, it has become apparent that DNA repair mechanisms are involved in the malignant progression and resistance to therapy of gliomas. Many investigators have shown that increased levels of O6-methyl guanine DNA alkyltransferase, a DNA monoalkyl adduct repair enzyme, are correlated with resistance of malignant glioma cell lines to nitrosourea-based chemotherapy. Three important DNA excision repair genes ERCC1 (excision repair cross complementation group 1), ERCC2 (excision repair cross complementation group 2), and ERCC6 (excision repair cross complementation group 6) have been studied in human tumors. Gene copy number variation of ERCC1 and ERCC2 has been observed in primary glioma tissues. A number of reports describing a relationship between ERCC1 gene alterations and resistance to anti-cancer drugs have been also described. The levels of ERCC1 gene expression, however, have not been correlated with drug resistance in gliomas. The expression of ERCC6 gene transcribes has been shown to vary with tissue types and to be highest in the brain. There have been no comprehensive studies so far, however, of ERCC6 gene expression and molecular alterations in malignant glioma. This project examined the ERCC1 expression levels and correlated them with cisplatin resistance in malignant glioma cell lines. We also examined the molecular alterations of ERCC6 gene in primary glioma tissues and cells and analyzed whether these alterations are related to tumor progression and chemotherapy resistance. Our results indicate the presence of mutations and/or deletions in exons II and V of the ERCC6 gene, and these alterations are more frequent in exon II. Furthermore, the mutations and/or deletions in exon II were shown to be associated with increased malignant grade of gliomas. The results on the Levels of ERCC1 gene transcripts showed that expression levels correlate with cisplatin resistance. The increase in ERCC1 mRNA induced by cisplatin could be down-regulated by cyclosporin A and herbimycin A. The results of this study are likely to provide useful information for clinical treatment of human gliomas. ^

LOSS OF GPRC5A ENHANCES SURVIVAL IN NORMAL AND MALIGNANT LUNG EPITHELIAL CELLS BY ELICITING PERSISTENT STAT3 ACTIVATION INDUCED BY AUTOCRINE LIF

Signal transduction and activator of transcription 3 (Stat3) is activated by cytokines and growth factors in many cancers. Persistent activation of Stat3 plays important role in cell growth, survival, and transformation through regulating its targeted genes. Previously, we found that mice with a deletion of the G protein-coupled receptor, family C, group 5, member a (Gprc5a) gene develop lung tumors indicating that Gprc5a is a tumor suppressor. In the present study, we examined he mechanism of Gprc5a-mediated tumor suppression. We found that epithelial cells from Gprc5a knockout mouse lung (Gprc5a-/- cells) survive better in vitro in medium deprived of exogenous growth factors and form more colonies in semi-solid medium than their counterparts from wildtype mice (Gprc5a+/+ cells). The phosphorylation of tyrosine 705 on Stat3 and the expression of Stat3-regulated anti-apoptotic genes Bcl-XL, Cryab, Hapa1a, and Mcl1 were higher in the Gprc5a-/- than in Gprc5a+/+ cells. In addition, their responses to Lif were different; Stat3 activation was persistent by Lif treatment in the Gprc5a-/- cells, but was transient in the Gprc5a+/+ cells. The persistent activation of Stat3 by Lif in Gprc5a-/- cells is due to a decreased level of Socs3 protein, a negative inhibitor of the Lif-Stat3 signaling. Restoration of Socs3 inhibited the persistent Stat3 activation in Gprc5a-/- cells. Lung adenocarcinoma cells isolated from Gprc5a-/- mice also exhibited autocrine Lif-mediated Stat3 activation. Treatment of Gprc5a-/- cells isolated from normal and tumor tissue with AG490, a Stat3 signaling inhibitor, or with dominant negative Stat3(Y705F) increased starvation-induced apoptosis and inhibited anchorage-independent growth. These results suggest that persistent Stat3 activation increased the survival and transformation of Gprc5a-/- lung cells. Thus, the tumor suppressive effects of Gprc5a are mediated, at least in part, by inhibition of Stat3 signaling through regulating the stability of the Socs3 protein.

Risk of Second Malignant Neoplasms Following Proton Arc Therapy and Volumetric Modulated Arc Therapy for Prostate Cancer

The risk of second malignant neoplasms (SMNs) following prostate radiotherapy is a concern due to the large population of survivors and decreasing age at diagnosis. It is known that parallel-opposed beam proton therapy carries a lower risk than photon IMRT. However, a comparison of SMN risk following proton and photon arc therapies has not previously been reported. The purpose of this study was to predict the ratio of excess relative risk (RRR) of SMN incidence following proton arc therapy to that after volumetric modulated arc therapy (VMAT). Additionally, we investigated the impact of margin size and the effect of risk-minimized proton beam weighting on predicted RRR. Physician-approved treatment plans were created for both modalities for three patients. Therapeutic dose was obtained with differential dose-volume histograms from the treatment planning system, and stray dose was estimated from the literature or calculated with Monte Carlo simulations. Then, various risk models were applied to the total dose. Additional treatment plans were also investigated with varying margin size and risk-minimized proton beam weighting. The mean RRR ranged from 0.74 to 0.99, depending on risk model. The additional treatment plans revealed that the RRR remained approximately constant with varying margin size, and that the predicted RRR was reduced by 12% using a risk-minimized proton arc therapy planning technique. In conclusion, proton arc therapy was found to provide an advantage over VMAT in regard to predicted risk of SMN following prostate radiotherapy. This advantage was independent of margin size and was amplified with risk-optimized proton beam weighting.

FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

Analysis of differential gene expression in nontumorigenic glioblastoma multiforme

Loss of chromosome 10 represents the most common cytogenetic abnormality in high grade gliomas (glioblastoma multiforme). To identify genes involved in the malignant progression of human gliomas, a subtractive hybridization was performed between a tumorigenic glioblastoma cell line (LG11) and a nontumorgenic hybrid cell (LG11.3) containing an introduced chromosome 10. LG11 mRNA was subtracted from LG11.3 cDNA to produce cDNA probes enriched for sequences whose expression differs quantitatively from the parental tumorigenic cells. Both known and novel sequences were identified as a result of the subtraction. Northern blot analysis was then used to confirm differential expression of several subtracted clones. One novel clone, clone 17, identified a 2.6 kb message that showed a consistent two to four fold increase in expression in the LG11.3 nontumorigenic cells. Clone 17 (340 bp) was used successfully to screen for a near full-length version, RIG (regulated in glioma), which was 2,569 bp in size. The RIG cDNA sequence showed homology to clone 17 and to an anonymous EST (IB666), but to no previously identified genes. This screening effort also identified several independent clones representing novel sequences, most of which failed to show increased expression in the nontumorigenic GBM cells. Tissue distribution studies of RIG indicated highest levels of expression in human brain with appreciably lower levels in heart and lung. In vitro transcription and translation experiments demonstrated the ability of RIG to direct the synthesis of a 13 kD protein product. However, open reading frame analysis revealed no identify with previously described motifs or any known proteins. Using a combination of somatic cell hybrid panels and in situ hybridization, the RIG gene was mapped to chromosome 11p14-11p15. Further study of RIG and related gene products may provide insight into the negative regulation of glial oncogenesis. ^

Relationships between the alterations of tumor suppressor genes, risk factors and clinical outcomes of gliomas

The relationship between MMAC/PTEN, DMBT1 and the progression and prognosis of glioma, and the association between the alterations of MMAC/PTEN, p53, p16, and Rb and some cancer risk factors, such as smoking, exposure to radiation, family cancer history, and previous cancer history, were assessed in 4 studies. ^ By allelic deletion analysis, MMAC/PTEN locus was shown to be frequently lost in glioblastomas multiforme (GM) but maintained in most lower-grade astrocytic tumors. DMBT1 locus, however, was frequently lost in all grades of gliomas examined. The potential biological significance of these two regions was frontier assessed by examining microcell-hybrids that contained various fragments of 10q. Somatic cell hybrid clones that retained the MMAC/PTEN locus have less transformed phenotypes, exhibiting an inability to grow in soft agarose. On the other hand, the presence or absence of DAMT1 did not correlate with any in vitro phenotype assessed in our model system. Further, Cox proportional hazards regression analysis, adjusted for age at surgery and histologic grades (GM, and non-GM), showed that without LOH at the MMAC/PTEN locus had a significantly better prognosis than did patients with LOH at MMAC/ PTEN (hazard ratio = 0.5; 95% Cl = 0.28–0.89; P = 0.018). Furthermore, status of LOH at MMAC/PTEN was found to be significantly associated with age, while that for DMBT1 was not. These results suggest that the DMBT1 may be involved early in the oncogenesis of gliomas, while alterations in the MMAC /PTEN may be a late event in the oncogenesis related with progression of gliomas and provide a significant prognostic marker for patient survival. ^ The associations between 4 cancer risk factors and 4 tumor suppressor genes were assessed. The expression of p16 was observed to be associated with current smoking (adjusted OR = 1.9, 95% CI = 1.02–3.6) but not the former smoking (adjusted OR = 1.1, 95% Cl = 0.5–3.5). The expression of p53 was found to be associated with the family cancer history (OR = 3.5, 95% Cl = 1.07–11 for patients with first-degree family history of cancer). MMAC/ PTEN was associated with the histologic grade (OR = 2.8, 95% CI = 1.2–6.6) and age (P = 0.035). Also, the OR for LOH around MMAC/PTEN in patients with a family history of cancer was elevated (OR = 1.9, 95% CI = 0.8–4.6 for patients with first-degree family history of cancer). The associations between exposure and the alterations of tumor suppressor genes, between smoking and p16, between family history of cancer and p53 and MMAC/PTEN, provide suggestive evidences that those exposures are related to the development of gliomas. ^

Mechanisms of Ad-p53 induced apoptosis in human malignant glioma

Malignant brain tumors are one of the most challenging cancers affecting society today. In a recent survey, an estimated 17,000 annual cases were recorded with a staggering total of 13,300 deaths. A unique degree of heterogeneity typifies glial tumors and presents a challenge for solitary anti-neoplastic treatments. Tumors subsist as heterogeneous masses that progress through dysplasia to astrocytomas, mixed glioma and glioblastoma multiforme. Although traditional therapeutic approaches have provided increments of success, the median survival time remains 12 months. The urgency to improve upon current clinical protocols has encouraged alternative experimental strategies such as p53 adenoviral gene therapy (Ad-p53). This study addresses the efficacy of Ad-p53 for the treatment of glioma. Our model presents a tumor response that is unique among human cancers. Ad-p53 effectively induces apoptosis in mutant p53 expressing cells yet fails to do so in those with wildtype p53. In order to adopt Adp53 as a standard anti-cancer modality, we characterized the role of the tumor suppressor gene p53 in mediating apoptosis. We demonstrate that altering cellular p53 status through the introduction of a dominant negative mutant p53 (175H, 248W, 273H) sensitized cells to Ad-p53. We discovered that wild-type p53 expressing glioma cells retain the apoptotic machinery necessary to accomplish cell death, but have developed mechanisms that interfere with p53 signaling. Earlier studies have not addressed the mechanisms of Ad-p53 apoptosis nor the resistance exhibited by wild-type p53 glioma. To explain the divergent phenotypes, we identified apoptotic pathways activated and effectors of the response. We illustrated that modulation of the death receptor Fas/APO-1 is a principal means of Ad-p53 signaling that is impaired in wild-type p53 glioma. Moreover, the apoptotic response was found to be a multi-faceted process that engaged several caspases, most notably caspases -1, -3 and -8. Lastly, we assessed the ability of anti-apoptotic molecules Bcl-2 and CrmA to inhibit Ad-p53 apoptosis. These studies revealed that Ad-p53 is a powerful tool for inducing apoptosis that can be delayed but not inhibited by anti-apoptotic means. This work is critical for understanding the development of glioma and the phenotypic and genotypic alterations that account for tumor resistance. ^